Cling-E. coli : Bacteria on target Try adding one single organizational slide to build upon as transitional slides between projects Harvard iGEM 2007 Ellenor Brown Stephanie Lo Alex Pickett Sammy Sambu Kevin Shee Perry Tsai Shaunak Vankudre George Xu

The motivation To develop a system for directing bacteria to a target of interest and effecting downstream activity How does this fit into iGEM and standard parts paradigm? What are your parts? Modularity? Simplify, simplify, simplify. Reader’s digest version of results. Too many words, more hard-hitting, crisper. Use headings and explain verbally. Suggest a broader, real-world application. Potential targets. Increasing local concentration improves the efficacy/toxicity margin. We may have steered away from standard BioBricks, but we’re going for more general application. Talk more about applications, pictures

Potential Targets and Applications Bind Proteins Bind Toxins Bind Viruses Bind Tissue Bind DNA Bind Other Cells Bind Surface

Fec signal transduction Quorum-sensing Fec signal transduction Bacterial targeting Quorum-sensing Fec signal transduction Organizational slide, transition to first topic of bacterial targeting Quorum-sensing Fec signal transduction

Surface Engineered Bacteria Engineered to Bind and Signal Fusion Protein OmpA – C terminal insertion OmpA-Loop1 insertion AIDA-1 – N terminal insertion FecA – loop insertion Membrane Protein Add a slide about the concept of the library.

Surface Engineered Bacteria Engineered to Bind and Signal Positive Signal Background AIDA-1 his or AIDA-1 strep2 Sender LuxI RFP Amp and Kan Kan Amp Co-transform signal

Selecting/enriching for surface engineered bacteria Direct Selection Direct magnetic beads Indirect selection MACS FACS

Direct Selection using Magnetic Beads After magnetic selection Convert tables to graphs/charts

Direct Selection using Magnetic Beads Convert tables to graphs/charts

Fec signal transduction Bacterial targeting Make grey darker Quorum-sensing Fec signal transduction

Cell-Cell Signaling: luxI/luxR Quorum Sensing Reporter Target (bead) R Receiver + OHHL Sender

Cell-Cell Signaling: Constructs Sender Receiver Single Cell Construct – “JT” Two Cell Construct Make a point that we used existing BioBricks successfully, and what parts we’ve built ourselves, bam. Also, split slides such that we show construct & fluorescence curves (both for two cell and single cell constructs) Voigt paper acknowledgement luxpR constructs Change blown-up pics to simpler Why use single-cell? Receiver Sender

Sharp increase in fluorescence indicates quorum activity Clarify what the assay was, possibly use a plot instead of bars.

Selection with Direct Magnetic Beads Control: no selection Experimental: Selection with beads

60-fold Enrichment through Direct Magnetic Beads Change “Experimental” to a more informative label & make the legend larger. Control: no beads Selection with streptactin beads

The plate-drop experiment BBa_T9002 BBa_S03623 – BBa_I13507 T9002 OHHL Receiver -> GFP S23I07 Red OHHL sender

Plate Drop Experiment with Enriched Sender

Fec signal transduction Bacterial targeting Too many words. Redraw diagram more simply or erase unnecessary parts. Maybe make an animation. Quorum-sensing Fec signal transduction

Direct Signaling from the Outer Membrane: the Fec System Advantages of Direct Signaling from the Outer Membrane: Substrate Specificity The FecIRA system is the only well-characterized signaling scaffold in Gram-negative bacteria FecA is an iron transporter and signal transducer on the outer membrane of E. Coli K-12 When ferric citrate binds, FecA activates periplasmic FecR, which then activates the sigma factor FecI, resulting in gene expression The system is repressed by the Fur repressor in iron-rich conditions Too many words. Redraw diagram more simply or erase unnecessary parts. Maybe make an animation. Braun et al. “Gene Regulation by Transmembrane Signaling.” Biometals 2006 Apr;19(2):103-13.

Fec: Motivation and Methods Structural information suggests possibility of maintaining signaling with changed binding. L7 moves up to 11Å, helix unwinds L8 moves up to 15Å Select binding targets by inserting random library, controls known to bind nickel and streptavidin into loops 7 and 8. Even if signaling cannot be maintained, binding of controls proves that FecA can be used as scaffold for surface expression of peptides Computational approach in collaboration with the lab of Costas Maranas, Penn State Dept of Chemical Engineering. Too many words. Point out areas on the structure more clearly. If there’re no results from collaboration, don’t mention it. Ferguson AD et al. “Structural Basis of Gating by the Outer Membrane Transporter FecA. Science 2002 Mar 1: 295(5560) 1715-9

Results Wild Type Induction of FecA with Sodium Citrate and a GFP Reporter shows approximately 2000 RFU increase MACS Results Results from Nickel and His Fluorescence Assays To be resulted

Biobricking the Fec System Construct Features: Swappable FecA - FecA is flanked by Nhe1 and AflII sites to allow the easy mutagenesis and replacement of FecA. Variable Promoters - each component will be on a separate constitutive promoter. The optimization of GFP expression using promoters of different strengths is planned. Conceptually good, but needs fewer words.

Biobricking the Fec System Mutagenesis of Fec promoter to weaken gene expression, providing a range of sensitivity. Mutagenesis of the Fec promoter to remove FUR repressor binding site, allowing easier assays. Studies detailing the response of reporter expression to mutations at each site of the promoter have been done. (Enz, Pressler, Braun papers)

CONCLUSION To be added Make a point of using surface expression and random libraries as a distinguishing factor.

ACKNOWLEDGEMENTS Advisors Teaching Fellows George Church Debra Auguste Jagesh V. Shah William Shih Pamela Silver Alain Viel Tamara Brenner Teaching Fellows Nicholas Guido Bill Senapedis Mike Strong Harris Wang